Methods and pharmaceutical compositions for treatment of cystic fibrosis

ABSTRACT

The present invention relates to a method and compositions for the treatment of cystic fibrosis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 15/021,137 filed on Mar. 10, 2016, now U.S. Pat. No. 9,688,990,which was a national stage filing under Rule 371 of internationalapplication PCT/EP2014/069522 filed Sep. 12, 2014, which itself claimedpriority to European Application 13306250.5 filed Sep. 12, 2013.

FIELD OF THE INVENTION

The present invention relates to a method and compositions for thetreatment of cystic fibrosis.

BACKGROUND OF THE INVENTION

Cystic fibrosis (CF) is the most common lethal monogenic disorder inCaucasians, with an incidence of one bird in 2500-4000 and 70,000affected people worldwide and kills most of them in their 20s. Cysticfibrosis is an autosomal recessive disease linked to mutations in thecystic fibrosis transmembrane conductance regulator gene (CFTR) genewhose nature determines the clinical expression and severity of thedisease, affecting mainly the respiratory, digestive and genitalsystems. CFTR, a chloride-ion channel, is involved in the changes ofsurface liquid covering airway epithelial cells. Dehydration of thesurface liquid leads to altered mucociliary clearance and inflammationand infections at the mucosal epithelia. CFTR mutations that reduce CFTRprotein function cause accumulation of thick, sticky mucus in thebronchi of the lungs, loss of exocrine pancreatic function, impairedintestinal secretion, and an increase in the concentration of chloridein the sweat (Boucher R C Trends Mol Med., 2007). In patients with CF,lack of CFTR Cl(−) channel function leads to progressive pulmonarydamage and ultimately to death. Chronic lung disease is the major causeof mortality and morbidity in CF patients.

Patients with CF require numerous therapies to manage these symptoms(Farrell P M et al. J Pediatr, 2008), including mucolytic and antibioticagents and chest physiotherapy to treat the airway disease and digestiveenzymes to replace the loss of exocrine pancreatic function. These andother interventions have increased life expectancy dramatically, butimprovement is needed to reduce the high treatment burden and increasesurvival (Sawicki G S et al. J Cyst Fibros, 2009). A CFTR potentiator(Ivacaftor) has been developed by Vertex Pharmaceuticals (Ramsey B W etal. N Engl J Med, 2011) and has been recently approved for the treatmentof CF patients carrying the p.Gly551Asp mutation (2-5% of all patients).To date, the drug has failed for CF patients with p.Phe508del, the mostcommon mutations. Several CFTR correctors have been previously reportedto be active in vitro (Hutt D M et al. Nature Chem Biol, 2010; Verkman AS and Galietta L J Nat Rev Drug Discov, 2009) but therapies for CF havenot yet advanced from these efforts. A corrector that corrects thetrafficking of the p.Phe508del protein is still under investigation inclinical trials (Van Goor F et al. PNAS, 2011).

Accordingly, there is a need to develop new drugs that will be suitablefor preventing or treating CF and CFTR-related diseases. In this way, ithas been suggested that characterization of new therapeutic compounds inCF and CFTR-related diseases may be highly desirable.

Since the cloning of the CFTR gene in 1989, nearly 2000 mutations of thegene have been described. The p.Phe508del mutation (deletion ofphenylalanine at position 508 of the protein) is the most frequent (70%of mutated alleles in patients with CF). This severe mutation impairsthe maturation of CFTR protein and the protein fails to reach cellmembrane. Other grouped according to their effect on the CFTR proteininclude mutations that result in a shortened protein, in a reducedchloride conductance, in a defective CFTR stability at the cell surfaceor in reduced numbers of CFTR transcripts due to incorrect splicing(Rowe S M et al. N Engl J Med, 2005).

Alternative splicing is a regulated process during gene expression thatresults in a single gene coding for multiple proteins, constitutingimportant post-transcriptional regulation of eukaryotic gene expression.In this process, particular exons of a gene may be included within, orexcluded from, the final, processed mRNA. Abnormal variations insplicing are also implicated in disease; a large proportion of humangenetic disorders result from splicing variants. Different CFTRmutations in splice sites or cis-acting splicing regulatory sites orresulting in the creation of new abnormal alternative donor or acceptorsite may lead to mis-splicing of multiple abnormal CFTR transcripts andnon functional CFTR proteins.

In addition to alternative splicing mechanisms, microRNAs (miRNA) canact in synchrony with transcription factors to control gene expression(Martinez N J et al. Bioessays, 2009; Shalgy R et al. Aging, 2009),revealing an important new complexity in the post-transcriptionalregulation of eukaryotic gene expression. Recently, it has been foundthat CFTR is post-transcriptionally regulated by miRNAs, such as miR-145and miR-494 (Gillen A E et al. Biochem J, 2011; Megiorni F et al. PlosOne, 2011; Ramachandran S et al. PNAS, 2012). Several miRNAs includingmiR-145 are expressed in primary human airway epithelial cells, whereCFTR expression is repressed (Gillen A E et al. Biochem J, 2011) or arederegulated in CF patients (Oglesby I K et al. J immunol, 2013;Ramachandran S et al. AJRCMB, 2013).

SUMMARY OF THE INVENTION

The present invention relates to methods and compositions for thetreatment of CFTR-related disease and cystic fibrosis.

DETAILED DESCRIPTION OF THE INVENTION

In the present invention and in the objective to identify new repressiveelements to design new tools for Cystic fibrosis (CF) correction, theinventors investigated the role of post-transcriptional regulation ofCFTR gene expression in CF. Through the identification of a molecularnetwork involving lung developmental-specific transcription factors andmiRNAs controlling CFTR gene expression, the inventors established theimportance of inhibitory motifs for the binding of regulators includingmiR-101, miR-145. By global profiling, the inventors also evidenced thatmiR-145 is one of the most deregulated microRNAs in adult versus fetallung. Identifying cis- and trans-repressors allowed the inventors toenvision new therapeutic tools for lung disease including cysticfibrosis (CF), and tested in reconstituted epithelia from nasal cells ofp.Phe508del CF patients and in CF bronchial epithelial cells. Afterdefining the regulatory motifs on the CFTR gene for the binding of themiRNAs of interest, the inventors then designed target-site blocker(TSB) (miRNA binding-blocker oligonculeotides (MBBO)) to prevent bindingof several miRNAs including miR-101, miR-600, miR-145 and miR-384 to the3′UTR of the CFTR gene.

The inventors have also investigated several mutations creating newabnormal alternative donor or acceptor site and then inducingalternative splicing resulting in abnormal CFTR transcripts and nonfunctional CFTR proteins. The inventors then designed target-siteblocker (TSB) targeting splice sites to prevent the binding ofspliceosome proteins to the alternative donor and acceptor sites of theCFTR transcripts.

Therefore, by using these specific TSB oligonucleotides, the inventorsdemonstrate the correction of the CFTR channel activity through eitheran increase of the CFTR transcripts and protein levels or therestoration of the CFTR full-length transcripts.

Oligonucleotides of the Invention

The present invention relates to isolated, synthetic or recombinantoligonucleotides recognizing or targeting CFTR mRNA.

The term “oligonucleotide” refers to isolated, synthetic or recombinantoligonucleotides recognizing or targeting CFTR mRNA. The term“oligonucleotide” also refers to antisense oligonucletotide (ASO) orblocker oligonucleotide (BO) or target-site blocker (TSB) recognizing ortargeting regulatory motifs on the CFTR mRNA. The term“oligonucleotides” also refers to miRNA binding-blocker oligonculeotides(MBBO) recognizing or targeting regulatory motifs on the CFTR mRNA toprevent binding of several miRNAs including miR-101, miR-600, miR-145and miR-384 to the 3′UTR of the CFTR mRNA. The term “oligonucleotide”also refers to splicing-blocker oligonucleotides (SBO) recognizing ortargeting splice sites to prevent the binding of spliceosome proteins tothe alternative donor and acceptor sites of the CFTR mRNA and thesplicing of a cryptic exon inserted into the mutant CFTR mRNA.

The term “CFTR” refers to cystic fibrosis transmembrane conductanceregulator, an ATP-binding cassette (ABC) transporter-class ion channelinvolved in the transport of chloride and thiocyanate ions acrossepithelial cell membranes. The term “CFTR” also refers to a chloride-ionchannel involved in the changes of surface liquid covering airwayepithelial cells.

The present invention also relates to an isolated, synthetic orrecombinant oligonucleotide comprising a nucleic acid sequence that hasat least about 90%, or at least about 95%, or at least about 96%, or atleast about 97%, or at least about 98%, or at least about 99% nucleicacid sequence identity with a sequence selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 andSEQ ID NO: 5.

Nucleic acid sequence identity is preferably determined using a suitablesequence alignment algorithm and default parameters, such as BLAST N(Karlin and Altschul, Proc. Natl Acad. Sci. USA 87(6):2264-2268 (1990)).

In a particular embodiment, the oligonucleotide according to theinvention comprises a nucleic acid sequence selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 andSEQ ID NO: 5.

In a particular embodiment, the oligonucleotide according to theinvention has a nucleic acid sequence selected from the group consistingof SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO:5.

The oligonucleotide of the invention may be of any suitable type. Theone skilled in the art can easily provide some modifications that willimprove the clinical efficacy of the oligonucleotide (C. Frank Bennettand Eric E. Swayze, RNA Targeting Therapeutics: Molecular Mechanisms ofAntisense Oligonucleotides as a Therapeutic PlatformAnnu. Rev.Pharmacol. Toxicol. 2010.50:259-293.). Typically, chemical modificationsinclude backbone modifications, heterocycle modifications, sugarmodifications, and conjugations strategies. For example theoligonucleotide may be selected from the group consisting ofoligodeoxyribonucleotides, oligoribonucleotides, LNA, oligonucleotide,morpholinos, tricyclo-DNA-antisense oligonucleotides (ASOs), U7- orU1-mediated ASOs or conjugate products thereof such aspeptide-conjugated or nanoparticle-complexed ASOs. Indeed, for use invivo, the oligonucleotide may be stabilized. A “stabilized”oligonucleotide refers to an oligonucleotide that is relativelyresistant to in vivo degradation (e.g. via an exo- or endo-nuclease).Stabilization can be a function of length or secondary structure. Inparticular, oligonucleotide stabilization can be accomplished viaphosphate backbone modifications.

In a particular embodiment, the oligonucleotide according to theinvention is a LNA oligonucleotide. As used herein, the term “LNA”(Locked Nucleic Acid) (or “LNA oligonucleotide”) refers to anoligonucleotide containing one or more bicyclic, tricyclic or polycyclicnucleoside analogues also referred to as LNA nucleotides and LNAanalogue nucleotides. LNA oligonucleotides, LNA nucleotides and LNAanalogue nucleotides are generally described in InternationalPublication No. WO 99/14226 and subsequent applications; InternationalPublication Nos. WO 00/56746, WO 00/56748, WO 00/66604, WO 01/25248, WO02/28875, WO 02/094250, WO 03/006475; U.S. Pat. Nos. 6,043,060,6,268,490, 6,770,748, 6,639,051, and U.S. Publication Nos. 2002/0125241,2003/0105309, 2003/0125241, 2002/0147332, 2004/0244840 and 2005/0203042,all of which are incorporated herein by reference. LNA oligonucleotidesand LNA analogue oligonucleotides are commercially available from, forexample, Proligo LLC, 6200 Lookout Road, Boulder, Colo. 80301 USA.

Other possible stabilizing modifications include phosphodiestermodifications, combinations of phosphodiester and phosphorothioatemodifications, methylphosphonate, methylphosphorothioate,phosphorodithioate, p-ethoxy, and combinations thereof. Chemicallystabilized, modified versions of the oligonucleotide also include“Morpholinos” (phosphorodiamidate morpholino oligomers, PMOs), 2′-O-Metoligomers, tricyclo (tc)-DNAs, U7 short nuclear (sn) RNAs, ortricyclo-DNA-oligoantisense molecules (U.S. Provisional PatentApplication Ser. No. 61/212,384 For: Tricyclo-DNA AntisenseOligonucleotides, Compositions and Methods for the Treatment of Disease,filed Apr. 10, 2009, the complete contents of which is herebyincorporated by reference).

Other forms of oligonucleotides of the present invention areoligonucleotide sequences coupled to small nuclear RNA molecules such asU1 or U7 in combination with a viral transfer method based on, but notlimited to, lentivirus or adeno-associated virus (Denti, M A, et al,2008; Goyenvalle, A, et al, 2004).

The oligonucleotide of the invention can be synthesized de novo usingany of a number of procedures well known in the art. For example, theb-cyanoethyl phosphoramidite method (Beaucage et al., 1981); nucleosideH-phosphonate method (Garegg et al., 1986; Froehler et al., 1986, Garegget al., 1986, Gaffney et al., 1988). These chemistries can be performedby a variety of automated nucleic acid synthesizers available in themarket. These nucleic acids may be referred to as synthetic nucleicacids. Alternatively, oligonucleotide can be produced on a large scalein plasmids (see Sambrook, et al., 1989). Oligonucleotide can beprepared from existing nucleic acid sequences using known techniques,such as those employing restriction enzymes, exonucleases orendonucleases. Oligonucleotide prepared in this manner may be referredto as isolated nucleic acids.

In a particular embodiment, the oligonucleotide of the present inventionis conjugated to a second molecule. Typically said second molecule isselected from the group consisting of aptamers, antibodies orpolypeptides. For example, the oligonucleotide of the present inventionmay be conjugated to a cell penetrating peptide. Cell penetratingpeptides are well known in the art and include for example the TATpeptide (Bechara C, Sagan S. Cell-penetrating peptides: 20 years later,where do we stand? FEBS Lett. 2013 Jun. 19; 587(12):1693-702). In aparticular embodiment, the second molecule is able to target theepithelial cell. In a particular embodiment, the molecule targets theCFTR transporter. Several antibodies, peptides and aptamers that bindwith high affinity to epithelial cell are described in Raksha J et al.Am J Respir Cell Mol Biol. 2010; Van Meegen M A et al. Plos One. 2011).

Therapeutic Methods and Uses of the Invention

The oligonucleotide of the invention may be used in a method ofpreventing or treating diseases in a subject in need thereof.

Therefore, a further aspect of the invention relates to theoligonucleotide of the invention for use as a medicament.

In one embodiment, the oligonucleotide according to the invention may beused in the prevention or treatment of CFTR-related disease in a subjectin need thereof.

The present invention also relates to the oligonucleotide according tothe invention for use in the prevention or treatment of cystic fibrosisin a subject in need thereof.

As used herein, the term “subject” denotes a mammal. In one embodimentof the invention, a subject according to the invention refers to anysubject (preferably human) afflicted or at risk to be afflicted withCFTR-related diseases. In a preferred embodiment of the invention, asubject according to the invention refers to any subject (preferablyhuman) afflicted or at risk to be afflicted with cystic fibrosis.

The method of the invention may be performed for any type ofCFTR-related disease such as pulmonary diseases, chronic obstructivepulmonary disease (COPD), lung cancer, Congenital absence of the vasdeferens (CAVD), Idiopathic chronic pancreatitis (ICP), bronchiectasis.In the context of the invention, CFTR-related disease can be diagnosedin a subject and determined in a biological sample with techniques thatare well known from the one skilled in the art. These methods include,without being limited, amplification methods such as quantitative PCR,CFTR gene sequencing, and splicing reporter assays.

The method of the invention may also be performed for any type of cysticfibrosis such as revised in the World Health Organisation Classificationof cystic fibrosis and selected from the E84 group: mucoviscidosis,Cystic fibrosis with pulmonary manifestations, Cystic fibrosis withintestinal manifestations and Cystic fibrosis with other manifestations.

In a particular embodiment, oligonucleotides of the invention may bedelivered in vivo alone or in association with a vector. In its broadestsense, a “vector” is any vehicle capable of facilitating the transfer ofthe oligonucleotide of the invention to the cells. Preferably, thevector transports the nucleic acid to cells with reduced degradationrelative to the extent of degradation that would result in the absenceof the vector. In general, the vectors useful in the invention include,but are not limited to, naked plasmids, non viral delivery systems(cationic transfection agents, liposomes, etc. . . . ), phagemids,viruses, other vehicles derived from viral or bacterial sources thathave been manipulated by the insertion or incorporation of theoligonucleotide sequences. Viral vectors are a preferred type of vectorand include, but are not limited to nucleic acid sequences from thefollowing viruses: RNA viruses such as a retrovirus (as for examplemoloney murine leukemia virus and lentiviral derived vectors), harveymurine sarcoma virus, murine mammary tumor virus, and rous sarcomavirus; adenovirus, adeno-associated virus; SV40-type viruses; polyomaviruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vacciniavirus; polio virus. One can readily employ other vectors not named butknown to the art. In a preferred embodiment, the oligonucleotidesequence is under the control of a heterologous regulatory region, e.g.,a heterologous promoter. The promoter can also be, e.g., a viralpromoter, such as CMV promoter or any synthetic promoters.

In a particular embodiment, two or even more oligonucleotides can alsobe used at the same time; this may be particularly interesting when theoligonucleotides are vectorized within an expression cassette (as forexample by U7 or U1 cassettes).

The present invention also relates to the oligonucleotide according tothe invention in combination with one or more anti-CFTR-related diseaseagent for use in the prevention or treatment of CFTR-related disease ina subject in need thereof.

The present invention also relates to the oligonucleotide according tothe invention in combination with one or more anti-cystic fibrosis agentfor use in the prevention or treatment of cystic fibrosis in a subjectin need thereof.

In one embodiment, the anti-cystic fibrosis agent or anti-CFTR-relateddisease agent may include a CFTR corrector or potentitator (such asivacaftor (VX-770, Kalydeco), VX-661, VX-809), osmotic agents (such asBronchitol), antioxidants drugs, modifier of mucus (such as Pulmozyme,Mucomyst), bronchodilatators (such as Ventolin, Serevent),anti-infective compounds (such as TOBI, Azithromycin, Josacine) orfurther anti-inflammatory drugs (such as Ibuprofen, Dexamethasone,Zyflo, Accolate).

The present invention also relates to a method for preventing ortreating CFTR-related disease in a subject in need thereof, comprisingthe step of administering to said subject the oligonucleotide accordingto the invention.

The present invention also relates to a method for preventing ortreating cystic fibrosis in a subject in need thereof, comprising thestep of administering to said subject the oligonucleotide of theinvention.

The present invention also relates to a method for preventing ortreating CFTR-related disease in a subject in need thereof, comprisingthe step of administering to said subject the oligonucleotide accordingto the invention in combination with one or more anti-CFTR-relateddisease agent.

The present invention also relates to a method for preventing ortreating cystic fibrosis in a subject in need thereof, comprising thestep of administering to said subject the oligonucleotide according tothe invention in combination with one or more anti-cystic fibrosisagent.

Pharmaceutical Compositions and Kits of the Invention

The oligonucleotide of the invention may be used or prepared in apharmaceutical composition.

In one embodiment, the invention relates to a pharmaceutical compositioncomprising the oligonucleotide of the invention and a pharmaceuticalacceptable carrier for use in the prevention or treatment ofCFTR-related disease in a subject in need thereof.

The present invention also relates to a pharmaceutical compositioncomprising the oligonucleotide of the invention and a pharmaceuticalacceptable carrier for use in the prevention or treatment of cysticfibrosis in a subject in need thereof.

Typically, the compound of the invention may be combined withpharmaceutically acceptable excipients, and optionally sustained-releasematrices, such as biodegradable polymers, to form therapeuticcompositions.

“Pharmaceutically” or “pharmaceutically acceptable” refer to molecularentities and compositions that do not produce an adverse, allergic orother untoward reaction when administered to a mammal, especially ahuman, as appropriate. A pharmaceutically acceptable carrier orexcipient refers to a non-toxic solid, semi-solid or liquid filler,diluent, encapsulating material or formulation auxiliary of any type.

In the pharmaceutical compositions of the present invention for oral,inhalation, sublingual, subcutaneous, intramuscular, intravenous,transdermal, local or rectal administration of the oligonucleotide,alone or in combination with another active principle, can beadministered in a unit administration form, as a mixture withconventional pharmaceutical supports, to animals and human beings.Suitable unit administration forms comprise oral-route forms such astablets, gel capsules, powders, granules and oral suspensions orsolutions, sublingual and buccal administration forms, inhalationadministration forms, aerosols, implants, subcutaneous, transdermal,topical, intraperitoneal, intramuscular, intravenous, subdermal,transdermal, intrathecal and nasal or intranasal administration formsand rectal administration forms.

Preferably, the pharmaceutical compositions contain vehicles which arepharmaceutically acceptable for a formulation capable of beingadministered by nasal administration or by inhalation. Nasaladministration may be under the form of liquid solution, suspension oremulsion. Solutions and suspensions are administered as drops. Solutionscan also be administered as a fine mist from a nasal spray bottle orfrom a nasal inhaler. Inhalation may be accomplished under the form ofsolutions, suspensions, and powders; these formulations are administeredvia an aerosol, droplets or a dry powder inhaler. The powders may beadministered with insufflators or puffers.

Pharmaceutical compositions of the present invention include apharmaceutically or physiologically acceptable carrier such as saline,sodium phosphate, etc. The compositions will generally be in the form ofa liquid, although this need not always be the case. Suitable carriers,excipients and diluents include lactose, dextrose, sucrose, sorbitol,mannitol, starches, gum acacia, calcium phosphates, alginate,tragacanth, gelatin, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, celluose, water syrup, methyl cellulose, methyland propylhydroxybenzoates, mineral oil, etc. The formulations can alsoinclude lubricating agents, wetting agents, emulsifying agents,preservatives, buffering agents, etc. Those of skill in the art willalso recognize that nucleic acids are often delivered in conjunctionwith lipids (e.g. cationic lipids or neutral lipids, or mixtures ofthese), frequently in the form of liposomes or other suitable micro- ornano-structured material (e.g. micelles, lipocomplexes, dendrimers,emulsions, cubic phases, etc.).

One skilled in the art will recognize that the amount of anoligonucleotide to be administered will be an amount that is sufficientto induce amelioration of unwanted disease symptoms. Such an amount mayvary inter alia depending on such factors as the gender, age, weight,overall physical condition, of the subject, etc. and may be determinedon a case by case basis. The amount may also vary according to the typeof condition being treated, and the other components of a treatmentprotocol (e.g. administration of other medicaments such as steroids,etc.). If a viral-based delivery of oligonucleotides is chosen, suitabledoses will depend on different factors such as the viral strain that isemployed, the route of delivery (intramuscular, intravenous,intra-arterial, oral, inhalation or other). Those of skill in the artwill recognize that such parameters are normally worked out duringclinical trials. In addition, treatment of the subject is usually not asingle event. Rather, the oligonucleotides of the invention will likelybe administered on multiple occasions, that may be, depending on theresults obtained, several days apart, several weeks apart, or severalmonths apart, or even several years apart.

Pharmaceutical compositions of the invention may include any furtheragent which is used in the prevention or treatment of cystic fibrosis orCFTR-related disease. For example, pharmaceutical compositions of theinvention can be co-administered with CFTR corrector or potentitator(such as ivacaftor VX-770, VX-661, VX-809, Kalydeco), osmotic agents(such as Bronchitol), antioxidants drugs, modifier of mucus (such asPulmozyme, Mucomyst), bronchodilatators (such as Ventolin, Serevent),anti-infective compounds (such as TOBI, Azithromycin, Josacine) orfurther anti-inflammatory drugs (such as Ibuprofen, Dexamethasone,Zyflo, Accolate).

The invention also provides kits comprising at least one oligonucleotideof the invention. Kits containing oligonucleotide of the invention finduse in therapeutic methods.

Oligonucleotide sequences SEQ ID NO: 1 for MBBO-1:AGT GAT ATT TTC TTA CAG TAA T SEQ ID NO: 2 for MBBO-2:ATA AAC CGC TGA AGT TTC CAG TTA TC SEQ ID NO: 3 for MBBO-3:ACA TTA TTA AAA TAA ATA TTT CCT AGA G SEQ ID NO: 4 for TSB1:GTT GGT ACT TCT GTA ATA SEQ ID NO: 5 for TSB2: ACC TTA CTT ATA TCT CAA

The invention will be further illustrated by the following figures andexamples. However, these examples and figures should not be interpretedin any way as limiting the scope of the present invention.

FIGURES

FIGS. 1A-1H:

Impact of miRNA-binding Blocker Oligonucleotides on CFTR Expression inPulmonary Cells.

(A) Impact of different MBBOs on the post-transcriptional regulation ofthe CFTR gene. MBBOs (100 nM) were transfected in A549 and Beas-2Bpulmonary cells. Luciferase activity data are normalized to miRCURY LNA™microRNA Inhibitor Negative Control A (control oligo). (B) Impact ofMBBO-1 on the endogenous CFTR transcript level in A549 and Beas-2Bpulmonary cells. CFTR mRNA level was assessed by RT-qPCR following thetransfection of MBBO-1 or the use of the control oligo. Data arenormalized to β-actin transcript level. (C) Impact of MBBO-1 on theendogenous CFTR transcript level in controls (n=8), or (D) in ALIepithelium cultured from p.Phe508del homozygous CF patients (n=6). CFTRmRNA level was assessed by RT-qPCR 24 h following 1, 3 or 5 treatmentswith MBBO-1 or the use of the control oligo. Data are normalized toβ-actin transcript level. (E) Impact of MBBO-1, MBBO-2 and MBBO-3 onendogenous CFTR transcript level in CF patients. Box plot was assessedon CFTR transcript level in p.Phe508del homozygous CF patients (24 hafter the first treatment with MBBO-1, MBBO-2, MBBO-3 or control oligo).Data are normalized to β-actin transcript level. (F) Impact of MBBO-1,MBBO-2 and MBBO-3 on endogenous CFTR protein level in CF patients.Immunoblots were performed with CFTR antibody (clone MM13-4) on totalprotein extract of p.Phe508del homozygous CF patients (24 h after thefirst treatment with MBBO-1, MBBO-2, MBBO-3 or control oligo). Data arenormalized to Lamin A/C protein level. In reconstituted airwayepithelium 100 nM of MBBOs were directly added to the apical side infree medium (without transfection reagent) for 2 hours in 37° C. CFTRactivity (G) in 16HBEo− (NCF) and CFBE (CF) cells and (H) in non-treated(NT) CFBE cells or MBBO-treated CFBE cells. Representative cellfluorescence recordings from bronchial cells transiently expressing thehalide-sensitive YFP (scale bar reports the percentage of total cellfluorescence). Extracellular addition of F (arrow) caused YFP quenchingwith a rate proportional to the rate of I⁻ influx and CFTR activity.Channel opening is detected by a decrease of the probe fluorescence. Theamount of quench is directly proportional to the Cl⁻ efflux. Graphrepresents the summary of data obtained from the functional assayreporting rates of I transport.

Values are extremely significant at *P<0.0001.

FIGS. 2A-2F: Correction of Aberrant Splicing in Bronchial Cells by TSB1.

A and C. RT-PCR analysis of total RNA from bronchial BEAS-2B cells wasperformed using specific primers to analyse splicing afterco-transfection of wild type (IVS12 wt) or mutant (c.1680-883A>G,c.1680-886A>G) minigenes and TSBs (WT, wildtype transcript and CEI,cryptic exon inclusion). A. Effect of TSB concentration on aberrantsplicing. Cells were transfected with different TSB1 concentrations (25,50, 100, 1000 nM) for 24 hours. C. Effect of incubation time (24 h, 48 hand 72 h) on splicing correction using 50 nM TSB1. TSB1 specificity wasconfirmed using a control TSB (CTL) at the different testedconcentrations and in combination with wild type and mutant minigenes;to avoid overloading the figure, only the assays at 50 nM or 24 h wereshown. B and D. Quantification of CEI and WT transcripts aftertransfection of TSBs at different concentrations (B) or at differenttime-points after transfection (D). RT-PCR was performed using aFluorescein amidite (FAM)-labelled forward primer located within thesplice donor exon and a reverse primer within the splice acceptor exonof the pSPL3 plasmid. The quantification, in percentage, was performedby dividing the area of the CEI peak by the area of all peaks(wild-type+CEI). Data correspond to the mean value of at least twoindependent experiments. E. RT-PCR analysis of total RNA was performedusing specific primers to analyse both transcripts (WT, wild-type andCEI, cryptic exon inclusion). F. TSB1 was transfected 24 h at 100 nM incombination with WT or mutant minigenes in nasal cells obtained from acontrol individual, cultured one week in BEGM (Lonza, Walkersville, Md.USA) and plated in 24-well plates. RT-PCR analysis of total RNA wasperformed from nasal cells using specific primers to analyse bothtranscripts (WT, wild-type and CEI, cryptic exon inclusion).

FIG. 3: Correction of Aberrant Splicing in Bronchial Cells by TSB2

Quantification of CEI and WT transcripts after co-transfection of TSB2at different concentrations and the c.1680-883A>G or c.1680-886A>Gminigenes. RT-PCR was performed using a Fluorescein amidite(FAM)-labelled forward primer located within the splice donor exon and areverse primer within the splice acceptor exon of the pSPL3 plasmid. Thequantification, in percentage, was performed by dividing the area of theCEI peak by the area of all peaks (wild-type+CEI). Data correspond tothe mean value of at least two independent experiments. TSB2 specificitywas confirmed by using a control TSB (CTL) at the different testedconcentrations and in combination with both WT and mutant minigenes; toavoid overloading the figure, only the assay at 50 nM is shown.

EXAMPLES Example 1

Material & Methods

Gene Reporter Vectors and Directed Mutagenesis

The 3′UTR of the CFTR gene (1.7 kb from the termination codon to thepoly-adenylation signal) was subcloned into the pGL3-Control vector(Promega) downstream of the Luciferase gene (pGL3C-CFTR-3UTR).Degeneration of cis-motifs was realized by direct mutagenesis using theQuickChange®II site-directed mutagenesis kit (Stratagene). Allconstructs were verified using direct sequencing.

microRNA Precursors and Target Site Blocker Oligonucleotides (TSB)

Pre-miR™ miRNA precursors and pre-miR™ miRNA precursor negative controlwere purchased from Ambion (Life Technologies). LNATm-enhancedoligonucleotides used as miRNA binding-blockers (MBBO) are named TSB,recognizing the CFTR 3′UTR overlapping the miR-101 (MBBO-1), miR-145(MBBO-2) and miR-384 (MBBO-3) target sites were designed (EXIQON). As acontrol, miRCURY LNA™ microRNA inhibitor negative control A was used(EXIQON).

Cell Culture

Human pulmonary epithelial cells (A549 and Beas-2B) were cultured aspreviously described (Gras D et al. J Allergy Clin Immunol 2012). Humanfetal bronchial epithelial cells (HBEpiC, ScienCell, Clinisciences),isolated from a fetus at around 22 weeks of pregnancy, were primarycultured in collagen I-coated cell culture flasks in bronchialepithelial cell medium (BEpiCM, ScienCell, Clinisciences). All cellswere cultured at 37° C. under 5% CO₂. Some studies were performed withhuman bronchial epithelial cell lines, the CFBE4lo−(p.PHE508del/p.PHE508del) and the wild-type CFTR expressing, 16HBE14o−cells provided by Dr. D. C. Gruenert (San Francisco, Calif., USA). Bothcell types were maintained in MEM with Earle's salts and 1-glutamine aspreviously described (Saint-Criq V et al. Eur J Pharmacol 2012), CellSignal, 2012).

Nasal cells from healthy or p.Phe508del homozygous CF individuals wereobtained (with the agreement No ID-RCB 2011-A01520-41 of the FrenchEthical Research Committee) by inferior turbinate epithelium scratchingwith ASI Rhino-Pro® Curette (Arlington Scientific). Bronchial EpithelialGrowth Medium (BEGM, LONZA) supplemented with antibiotics (LifeTechnologies) was used to promote epithelial cells initial proliferationon collagen I-coated flasks. After three weeks of monolayer growth in37° C. 5% CO₂ atmosphere, cells were plated at 300,000 cells incollagen-coated 12 mm Transwell-Clear® support, 0.4 μm pore size,(Corning, Inc.). ALI (air-Liquide interface) medium was used to supportgrowth and epithelial differentiation. Top ALI medium (50:50 BEGM andDMEM 1 g/L Glucose) supplemented with specific additives (LONZA) plusPenicillin, Streptomycin and Amphotericin B (1×) (Life Technologies) wasremoved after confluence and bottom ALI medium was changed every 2-3days. Experiments were performed when epithelia were well-differentiated(at least 28 days).

Transient Transfections

For Luciferase assays, cells seeded at a density of 10,000 (Beas-2B),13,000 (A549) and 20,000 (HBEpiC) cells/100 μl of medium in 96-wellplates and were transfected with 72 ng of indicated reporter vector, and8 ng of internal control pRL-SV40 containing Renilla Luciferase(Promega) to normalize for transfection efficiency, by using Fugene®6transfection reagent (Roche Applied Sciences). Co-transfectionexperiments with miRNA or TSBs (MMBOs) were performed using Interferin®transfection reagent (Polyplus, Ozyme). In addition to the reportervector and pRL-SV40, 20 nM of microRNA precursors or 100 nM of TSBs(MMBOs) were co-transfected.

For RNA and protein studies, cells were seeded at a density of 250,000(Beas-2B), 300,000 (A549), 500,000 (HBEpiC) cells/2 ml of medium in6-well plates. Cells were also transfected with the same amount of miRNAprecursors or TSBs (MMBOs) as defined above.

RNA Extraction, Reverse Transcription and Quantitative PCR (qPCR)

Total RNAs were extracted, reverse transcribed and amplified aspreviously reported (Viart V et al. Eur J Hum Genet 2012), with minormodifications. Reverse transcription was performed with either randomprimers or CFTR and β-actin specific primers. Quantitative PCR wascarried out with 1:10 diluted cDNA and amplified with the LightCycler®480 SYBR Green I Master (Roche Applied Science). Relative expressionlevels were calculated using the comparative DDCt method with ahousekeeping gene (β-actin) expression as the endogenous control. miRNAswere purified with the miRNeasy Mini Kit and RNeasy MinElute Cleanup kit(Qiagen). Reverse transcription was performed on 40 ng of miRNA with themiRCURY LNA™ Universal cDNA synthesis kit (EXIQON) and qPCR wasperformed with 1:10 diluted cDNA and microRNA LNA™ primer set, specificfor each miRNA (EXIQON). Relative expression levels were calculatedusing the comparative DDCt method with SNORD44 and SNORD48 smallnucleolar RNA as the endogenous controls. To validate key microarrayresults (miRNA and RNA profiling), RT-qPCR was performed as explainedabove.

Reporter Assay

Cells were harvested 48 h after transfection, and the activity ofFirefly Luciferase and Renilla Luciferase was measured using theDual-Glo® Luciferase Assay System (Promega).

Western Blot

Whole proteins were directly extracted using 1× Laemmli buffer. Proteinswere separated on 7% or 10% SDS-PAGE gels and transferred to PVDFmembrane (Westran® Clear Signal Whatman®, Dutsher SAS). The proteinswere detected using 1:400 diluted anti-CFTR (clone MM13-4, Millipore) in5% skim milk. The protein levels of Lamin A/C (1:10,000, Sigma Aldrich)were assayed for internal control of protein loading. Following 1 h ofincubation with the anti-mouse secondary antibodies, proteins wererevealed by chemiluminescence.

CFTR Activity

The activity of CFTR protein was assessed by I− quenching ofhalide-sensitive YFP as previously described (Saint-Criq V et al. Eur JPharmacol 2012) using Premo Halide sensor technology (Invitrogen,Villebon sur Yvette, France). Forty hours following MBBO treatment, CFTRconductance was stimulated by an agonist mixture (forskolin,3-isobutyl-1methylxanthine, apigenin). After 10 min, the 96-well plateswere transferred to a plate reader for fluorescence assay. Each well wasassayed individually for CFTR-mediated I⁻ efflux by recordingfluorescence continuously (400 ms/point) for 2 s (base line), then 50 μlof a 140 mM I⁻ solution.

Statistical Analysis

For Luciferase and RT-qPCR assays, experiments were performed at leastthree times with samples analyzed at least in triplicate and pairedcomparisons were made using Student's t-test using InStat (GraphPadSoftware, version 3.0, Instat 3 folder). Data were expressed as themean±SE and were considered statistically significant at p<0.0001. Forassessing MBBO impact, statistical analyses were made using Wilcoxonstatistics with R software that generates box plots with significances.

Results

A Complex Pattern of Cis and Trans-acting Elements in the 3′UTR isInvolved in the Regulation of the Temporal Expression of CFTR Gene

To evaluate the effect of the 3′UTR on the post-transcriptionalregulation of the CFTR gene, the inventors transfected adult cell linesand primary fetal HBEpiC with a reporter vector, either with or withoutthe CFTR 3′UTR. The results show that the 3′UTR of the CFTR gene inducedstrong repression of Luciferase activity, in all cell-types, indicatingthat this region contains cis-repressive elements. Using thebioinformatic tool AREsite(http://rna.tbi.univie.ac.at/cgi-bin/AREsite.cgi), the inventorsidentified four putative AU-rich elements (ARE) in the 3′UTR of the CFTRgene: ARE-4816, ARE-5533, ARE-5698 and ARE-6074. These sites areadditional to those previously described (Baudouin-Legros M et al.AJPCP, 2005), that the inventors renamed ARE-4585, ARE-4760 and ARE-4891according to their nucleotide position. The inventors next evaluated thedegeneration of these motifs using reporter assays. Only one motif,ARE-4760, appeared to be implicated in mRNA stabilization, as itsdegeneration was associated with a decrease in Luciferase activity.Although ARE-4585, ARE-5533, ARE-5698 and ARE-6074 appeared to beinvolved in destabilization in A549 and Beas-2B, they had no effect inHBEpiC. The strongest effect was obtained using ARE-5698, identified insilico as the most conserved ARE motif in the CFTR 3′UTR. Othercis-acting elements might explain the repressive activity of the 3′UTRof CFTR in adult cell lines. Computational predictions using TargetScan(http://www.targetscan.org/), Pictar (http://pictar.mdc-berlin.de),miRanda (http://www.microrna.org/microrna/home.do) and miRDB(http://mirdb.org/miRDB/) detect thirteen putative miRNA binding motifsin the CFTR 3′UTR. Of the eight miRNAs previously studied, miR-145 hasbeen involved in the regulation of CFTR expression in colonic andpancreatic cell lines (Gillen A E et al. Biochem J, 2011).

The inventors then assessed the role of miRNAs in thepost-transcriptional control of CFTR in pulmonary cells by usingLuciferase reporter assays after transfection with precursors. MiR-665,miR-383 and miR-1290 did not induce an effect in any cell type, whereasmiR-600 affected Luciferase activity in all cells studied. MiR-505,miR-943, miR-377, miR-145, miR-384, miR-101 and miR-1246 only induced adecrease in Luciferase activity in A549 and/or Beas-2B, but not inHBEpiC. To confirm the importance of the cognate cis-elements, theinventors degenerated the motif for the binding of both miR-505 andmiR-101 resulting in the greatest effect on CFTR post-transcriptionalregulation. Two such degenerated sites were associated with a modestincrease of Luciferase activity, but only in adult pulmonary cells. Theinventors next validated the repressive effect of miR-101 onpost-transcriptional regulation of the CFTR gene in adult pulmonarycells. Overexpression of the miR-101 precursor was controlled.

Previous studies have demonstrated that miRNA-mediated regulation mightrequire the presence of an ARE sequence (Jing Q et al. Cell, 2005; Sun Get al NAR, 2010; Glorian V et al. Cell Death Differ, 2011). Both themiR-101 and miR-600 binding sites overlap the ARE-6074 motif and themiR-384 binding site overlaps the ARE-5698 motif. To investigate whetherthe impact of the miRNAs binding is dependent on the integrity of AREmotifs, we performed co-transfection assays with the miR-101, miR-600and miR-384 precursors and the constructs containing the wild-type ordegenerated CFTR 3′UTR. Only miR-101 lost its repressive effect onLuciferase activity following both the degeneration of the CFTR sequencehomologous to its seed region and abrogation of ARE-6074. Degenerationof ARE-6074 and ARE-5698 did not affect the activity of miR-600 andmiR-384, respectively.

These data demonstrate the implication of miRNAs in the tightlycontrolled developmental regulation of CFTR expression and moreparticularly that miR-101 directly acts on its cognate site incombination with an overlapping ARE motif.

From Identifying Crucial Regulators to Testing New Potential TherapeuticTools for Cystic Fibrosis

The region encompassing the miR-101 binding site and ARE-6074 iscritical to the regulatory action of miR-101. Thus, miRNAbinding-blocker oligonucleotides (MBBO) were designed to prevent thebinding of several miRNAs including miR-101, miR-600, miR-145 andmiR-384 to the 3′UTR of the CFTR gene. Use of these MBBOs,co-transfected with the reporter gene, led to a 1.5- to 6-foldoverexpression of Luciferase activity in different cell lines (FIG. 1A).The increase in level of endogenous CFTR by the MBBO-1 was confirmed inpulmonary cells (FIG. 1B).

Next, in order to evaluate the effect of the MBBOs in vivo, theinventors introduced MBBOs into reconstituted ALI epithelium culturedfrom human nasal cells from control individuals and p.Phe508delhomozygous CF patients. Control oligonucleotide, MBBO-1 or MBBO-2 wasadded to the apical side of primary nasal cells without transfectionreagent. After 2 h at 37° C., the apical medium was removed to restorethe air-liquid interface. Application was repeated every 2 days witheither freshly prepared control oligonucleotide or MBBOs. Use of MBBO-1induced a 2- to 6-fold increase of the endogenous CFTR transcript levelin controls (FIG. 1C) and a 2- to 3-fold increase in patients (FIG. 1D),and no marked improvement with a repeated number of incorporation of theMBBO-1. FIG. 1E represents the box plot of the MBBO-1, MBBO-2 and MBBO-324 h post-treatment in ALI epithelium cultured from p.Phe508delhomozygous CF patients (FIG. 1E). Immunoblot assays also revealed astronger expression of CFTR proteins in epithelium treated with both theMBBO-1, MBBO-2 and MBBO-3 in ALI epithelium cultured from CF patients(FIG. 1F). Functional assays showed the absence of CFTR-dependent aniontransport in CFBE41o-cells compared to wild-type 16HBEo− (FIG. 1G). Incontrast, a significant increase in level of anion transport wasobserved in CF cells treated with MBBO compared to the non-treated CFBEin accordance with the functional CFTR amount detected by immunoblot(FIG. 1H).

These data support the importance of the regions encompassing themiR-101 and miR-145 binding sites in regulation of the CFTR gene innative cells and offer a new insight for CF therapeutics.

Discussion

Herein, the inventors showed that miRNAs, including miR-101 and miR-145,negatively regulate the level of CFTR transcripts in adult lung cellswhilst having no effect in fetal lung cells. Interestingly, in additionto its specific role in mature lung cells, miR-101 has recently beendescribed as not altering CFTR mRNA stability in pancreatic cell linesGillen A E et al. Biochem J, 2011) but inducing a decrease of Luciferaseactivity in an embryonic kidney cell line (Mergiorni F et al Plos One,2011), suggesting a potential role as a tissue-specific factor. Theinventors demonstrated the implication of miRNAs, in the tightlycontrolled developmental regulation of CFTR expression and moreparticularly, that miR-101 acts on its cognate site in combination withan overlapping ARE motif.

Herein, the inventors also demonstrated the benefit of characterizingregulatory factors to identify novel therapeutic targets. Additionally,early studies indicated that complementation of as few as 6-10% CFTRtranscripts generate enough CFTR levels to maintain normal chloridetransport in the epithelia (Sinn P L et al. Hum Mol Genet, 2011). Thesedata are supported by findings that the presence of a naturallyoccurring sequence variation in the CFTR promoter, in cis of a severemutation, which increases transcription, can allow enough CFTR proteinto reach apical membrane cells in order to restore partial function,thus inducing a moderate CF phenotype (Romey M C et al. JBC, 2000). Morerecently, increasing the amount of p.Phe508del CFTR protein has beenassociated with an activated p.Phe508del CFTR channel activity (Hutt D Met al. Nat Chem Biol, 2010). A recent work demonstrates that use ofmiR-138 mimic as potential therapeutic tool, restores CFTR-Phe508del anda functional Cl− transport (Ramachandran S et al. PNAS, 2012). AsmiR-138 targets SIN3, a highly conserved transcriptional repressorregulating many genes, the authors underlined that use of miR-138 mimicmay have undesirable effect (Ramachandran S et al. PNAS, 2012). Herein,the inventors tested a new putative therapeutic tool that specificallytargets the CFTR gene. Focusing on miR-101 and miR-145, the inventorsdesigned MBBO oligonucleotides, recognizing their binding sites in theCFTR 3′UTR. This blockage led to the correction of the CFTR channelactivity through increase of mRNA and protein levels in CF patients withthe most severe mutation, the p.Phe508del in homozygous. As miR-101 andmiR-145 knock-down is associated with the dysregulation of epigeneticpathways resulting in cancer progression (Varambally S et al. Science,2008) and lung cancer (Guan P et al. J Exp Clin Cancer Res, 2012),inventor's approach of blocking their binding to their cognate CFTR mRNAmotif may have therapeutic benefits by stabilizing CFTR transcripts,ultimately providing enough functional proteins to improve CF patientsphenotype.

Example 2

Material & Methods

TSB Oligonucleotides sequences TSB1: GTT GGT ACT TCT GTA ATA TSB2:ACC TTA CTT ATA TCT CAA

In Silico Analysis

First, the CFTR variants was analysed using Human Splicing Finder 2.4.1(http://www.umd.be/HSF/HSF.html), including two different calculationalgorithms (HSF and MaxEnt) and NNSplice(http://www.fruitfly.org/seq_tools/splice.html; Splice Site Predictionby Neural Network or SSPNN). We assumed that aberrant splicing couldoccur when a de novo splice site (ss) was predicted or when the score ofa sub-optimal pre-existing ss was dramatically increased in the mutatedsequence. We also evaluated wild type (wt) and mutated sequences usingthe SpliceAid 2 database (http://193.206.120.249/splicing_tissue.html).

Second, we evaluated the conservation of the nucleotide positionaffected by a substitution in a set of selected mammalian orthologues.We first aligned the human genomic sequence of CFTR (NC_000007.13) withnine vertebrate orthologues from Ensembl using the MUSCLE3.7 algorithmavailable at www.phylogeny.fr. Results, shown as percentages of the wtand variant nucleotides, were obtained by analysing the multiplealignments with the Jalview software (http://www.jalview.org/). Abroader analysis with visualization of the multiple alignments of 19CFTR orthologues, including human CFTR, was obtained from UCSC using theMultiz Alignments tool. The wt nucleotide was considered as highlyconserved when its frequency was higher than 90%, intermediatelyconserved between 50 and 90% and poorly conserved when its frequency waslower than 50% and/or when the mutated nucleotide was found with afrequency of at least 10%.

Splicing Reporter Constructs

The impact of the newly discovered variant on splicing was tested usingthe pSPL3 exon-trapping vector (kindly provided by Dr I. Bottillo). Weamplified the CFTR sequence of interest (632 bp) in intron 12 (legacynomenclature: intron 11) using the patient's genomic DNA (diluted to 5ng/μl) and the High Fidelity Phusion® polymerase (Finnzymes, Espoo,Finland). The amplicon was inserted in pSPL3 between the XhoI and NheIrestriction sites using the T4 DNA ligase High Concentration(Invitrogen, Villebon sur Yvette, France) according to themanufacturer's instructions. In addition to the wild-type (wt) andmutated (c.1680-883A>G) minigenes, two other CFTR minigenes weregenerated carrying the SNP c.1680-870T>A (negative control for aberrantsplicing) or the splicing mutation c.1680-886A>G (1811+1.6kb A>G)(positive control for aberrant splicing). All primer sequences areavailable upon request. The sequences of the minigene constructs wereverified by Sanger sequencing.

Cell Culture, Transfection and Target Site Blocker (TSB) Treatment

Human bronchial BEAS-2B cells were cultured as previously described(Rene C et al. Cell. Mol. Life Sci. 2010). Twenty-four hours beforetransfection, cells were plated in six-well plates and, once at about80% confluence, transiently transfected with 1.5 μg of each minigeneconstruct using the PolyFect® transfection reagent (Qiagen, Courtaboeuf,France). Cells were harvested after 48 hours for transcript analysis.For TSB (TSB1 and TSB2) treatment, cells were co-transfected with theCFTR minigene constructs and 25 nM, 50 nM, 100 nM or 1 μM TSB using theInterferin® transfection reagent (Polyplus, Ozyme, Illkirch, France).

Transcript Analysis

Total RNA was extracted from BEAS-2B cells using the RNeasy Plus kit(Qiagen). At least, two independent transfections were carried out forall experimental conditions. Impact on splicing was tested as previouslyreported. The RT-PCR products were also sequenced using the Big DyeTerminator v1.1 Cycle Sequencing Kit (Applied Biosystems) on anABI-3130XL Genetic Analyzer. The relative amount of each CFTR splicingproduct was determined by measuring the peak area (evaluated by theGeneMapper software) and dividing it by the sum of all peak areasdetected in the same PCR reaction.

Total RNA was extracted from the patient's nasal cells and from twonon-CF controls using the RNeasy Plus kit (Qiagen). Reversetranscription was produced from 500 ng of total RNA with the MMLV-RT(Invitrogen). One μL of each RT-PCR was used for PCR amplification withprimers encompassing intron 12 (f11-r13) and specific primers pairsamplifying pseudoexon, PE (f11-rPE).

Results

Identification of a New Putative Disease-causing Mutation

To explore the effect of the c.1680-883A>G variation (located in intron12; chromosome location: 117,229,524; hg19), donor (5′ss) and acceptorsite (3′ss) in silico predictions were generated for the mutatedsequence. The mutation generated a new, high-score 5′ss in intron 12,suggesting that this site could be used for alternative splicing. Thenewly identified putative disease-causing mutation c.1680-883A>G isthree nucleotides away from a well-known splice mutation [c.1680-886A>G(1811+1,6 kbA>G) Chillon M et al. Am J Hum Genet, 1995] that creates adonor site causing the inclusion of a PE in mature transcripts. In otherrespects, the mutation was tested in 200 control chromosomes by Sangersequencing analysis and was not found.

Confirmation of the c.1680-883A>G Intronic Mutation Using a SplicingReporter Assay and in Nasal Cells of a Patient

We next used a splicing reporter assay to test the impact of thec.1680-883A>G variant on splicing. When BEAS-2B cells were transfectedwith the minigene carrying the c.1680-886A>G variant (used as positivecontrol), which causes the inclusion of an intronic sequence of 49 bp(cryptic exon inclusion, CEI), aberrantly-spliced transcripts wereapproximately 90-95% of the total (wild-type+aberrantly-spliced) CFTRmRNA. Conversely, transfection of the minigene carrying the neutralvariant c.1680-870T>A (negative control) did not have any effect onsplicing. Finally, transfection of the minigene carrying the newlyidentified c.1680-883A>G mutation led to activation of a pseudoexon (PE)resulting in the inclusion of an additional sequence of 53 bp, as shownby Sanger sequencing, and complete loss of wt CFTR transcripts.

We next checked if this mutation induced the sequence retention in nasalcells from the CF patient included in the family trio analysis. Thus, weconfirmed that the patient harboured a PE inclusion of 53 bp in intron12 by PCR amplification compared to controls using non-specific andspecific primers pairs to the PE (f11-r13 and f11-rPE, respectively).

Correction of CFTR Aberrant Splicing by Using Target Site Blockers (TSB1and TSB2)

We designed anti-sense oligonucleotides (TSB1 and TSB2) that blockaccess to the 3′ss (acceptor site) and 5′ss (donor site) respectively,in order to correct aberrant splicing caused by the c.1680-883A>G andc.1680-886A>G mutations. To determine the effect of TSB concentration onaberrant splicing, human bronchial BEAS-2B cells were co-transfectedwith the minigenes harbouring the two mutations and four different TSBconcentrations (25 nM, 50 nM, 100 nM, 1 μM) for 24 h. TSB1, whichtargets the 3′ss (acceptor site), had a marked corrective effect at lowconcentration (50 nM) on aberrant splicing caused by the c.1680-883A>Gand c.1680-886A>G mutations (FIG. 2A, upper and lower panel,respectively). TSB1 specificity was confirmed by using a TSB control(CTL). The efficiency of wt splicing restoration was quantified byfragment analysis PCR (FIG. 2B). We next performed time courseexperiments by transfecting 50 nM TSB1 and harvesting cells after 24 h,48 h and 72 h. A marked effect was evident already after 24 h (FIG. 2C).Specifically, quantification showed that the percentage of aberrantlyspliced transcripts (containing the cryptic exon) was reduced to 45%(c.1680-883A>G) and to 30% (c.1680-886A>G) of the total CFTR mRNA(wild-type+aberrantly-spliced transcripts). Thus, transfection of 50 nMTSB1 for 24 h induced a restoration of 55% and 70% of normal CFTR mRNA,respectively (FIG. 2D). Finally, we assessed the duration of action ofboth TSB (TSB1 and TSB2) in BEAS-2B cells and found that they had astrong effect on splicing up to 72 h after washing off the transfectionmedium (16 h incubation) (FIG. 2E). Partial restoration ofcorrectly-spliced CFTR mRNA induced by TSB1 (24 h at 100 nM) wasconfirmed in primary nasal cultures obtained from a control individu(FIG. 2F). TSB2 required a higher concentration for acting on splicing(FIG. 3).

Discussion

Among the 1976 reported CFTR mutations, 228 (11.54%) are believed toaffect pre-mRNA splicing (www.genet.sickkids.on.ca/). Most splicingmutations disrupt the canonical splice-site sequences, completelyabolishing exon recognition and/or leading to a nearly complete absenceof correctly spliced transcripts. Currently, 2 to 5% of CF mutationsremain unknown and are probably deeply located in introns, inducingaberrant splicing events. Functional analysis, minigene splicing assayand PCR on nasal cells from a CF patient carrying c.1680-883A>G, showedthat this deep intronic mutation generated a new, high-score 5′ ss(donor site) in intron 12 that is involved in PE inclusion.Interestingly, this mutation is close to another previously identifieddeep intronic mutation, the c.1680-886A>G, that also inducespseudoexonPE inclusion, suggesting that this intronic region may beprone to mutation. The c.1680-886A>G mutation occurs with a frequency of3.4% in the South-West part of Europe and of 0.2% in France (Federici S,2001). Conversely, c.1680-883A>G has never been described before, thoughhere identified in three unrelated patients.

The final objective of this work was the design of antisenseoligonucleotides for CF treatment. Indeed, PE exclusion by antisensemodification of pre-mRNA splicing represents a type of personalizedgenetic medicine. The development of oligonucleotides that block accessto a target site (Target site blockers, TSB) offers new treatmentopportunities for other genetic disorders (Webb T R et al. Hum Mol Genet2012, Nuzzo F et al. Blood, 2013). Here, we used this approach tocorrect the aberrant splicing caused by deep intronic mutations in theCFTR gene (c.1680-883A>G and c.1680-886A>G). TSB effect on aberrantsplicing correction in bronchial BEAS-2B cells was rapid and maintainedover time, suggesting that TSBs could be a therapeutic tool in patientswith CF who have deep intronic mutations in the CFTR gene because theyrestore normal transcripts. For patients with CF, these data areparticularly interesting because the c.1680-886A>G mutation is thefourth most frequent in South-West Europe (3.4%) and the threshold offunctional mRNA and subsequently of CFTR protein required for normalfunctions is very low, having been estimated at 5% (Ramalho A S et al.Am J Respir Cell Mol Biol 2002). It would be interesting, if possible,to test these TSBs in airway cells from patients with CF harbouring bothmutations tested in this work and also to assess TSBs for other intronicsplicing mutations in CFTR.

REFERENCES

Throughout this application, various references describe the state ofthe art to which this invention pertains. The disclosures of thesereferences are hereby incorporated by reference into the presentdisclosure.

Baudouin-Legros M, Hinzpeter A, Jaulmes A, Brouillard F, Costes B, etal. (2005) Cell-specific posttranscriptional regulation of CFTR geneexpression via influence of MAPK cascades on 3′UTR part of transcripts.Am J Physiol Cell Physiol 289: C1240-1250.

Boucher R C. Cystic fibrosis: a disease of vulnerability to airwaysurface dehydration. Trends Mol Med. 2007 June; 13(6):231-40.

Chillon M, Dork T, Casals T, et al. A novel donor splice site in intron11 of the CFTR gene, created by mutation 1811+1.6 kbA->G, produces a newexon: high frequency in Spanish cystic fibrosis chromosomes andassociation with severe phenotype. Am J Hum Genet 1995; 56:623-9

Farrell P M, Rosenstein B J, White T B, Accurso F J, Castellani C,Cutting G R, Durie P R, Legrys V A, Massie J, Parad R B, Rock M J,Campbell P W 3rd; Cystic Fibrosis Foundation. Guidelines for diagnosisof cystic fibrosis in newborns through older adults: Cystic FibrosisFoundation consensus report. J Pediatr. 2008 August; 153(2):S4-S14.

Federici S, Iron A, Reboul M P, et al. [CFTR gene analyis in 207patients with cystic fibrosis in southwest France: high frequency ofN1303K and 1811+1.6bA>G mutations]. Archives de pediatrie: organeofficiel de la Societe francaise de pediatrie 2001; 8:150-7.

Gillen A E, Gosalia N, Leir S H, Harris A (2011) MicroRNA regulation ofexpression of the cystic fibrosis transmembrane conductance regulatorgene. Biochem J 438: 25-32. Jing Q, Huang S, Guth S, Zarubin T, MotoyamaA, et al. (2005) Involvement of microRNA in AU-rich element-mediatedmRNA instability. Cell 120: 623-634.

Glorian V, Maillot G, Poles S, Iacovoni J S, Favre G, et al. (2011)HuR-dependent loading of miRNA RISC to the mRNA encoding the Ras-relatedsmall GTPase RhoB controls its translation during UV-induced apoptosis.Cell Death Differ 18: 1692-1701.

Gras D, Bourdin A, Vachier I, et al. An ex vivo model of severe asthmausing reconstituted human bronchial epithelium. J Allergy Clin Immunol2012; 129: 1259-1266.

Guan P, Yin Z, Li X, Wu W, Zhou B (2012) Meta-analysis of human lungcancer microRNA expression profiling studies comparing cancer tissueswith normal tissues. J Exp Clin Cancer Res 31: 54.

Hutt D M, Herman D, Rodrigues A P, Noel S, Pilewski J M, Matteson J,Hoch B, Kellner W, Kelly J W, Schmidt A, Thomas P J, Matsumura Y, SkachW R, Gentzsch M, Riordan J R, Sorscher E J, Okiyoneda T, Yates J R 3rd,Lukacs G L, Frizzell R A, Manning G, Gottesfeld J M, Balch W E. Reducedhistone deacetylase 7 activity restores function to misfolded CFTR incystic fibrosis. Nat Chem Biol. 2010 January; 6(1):25-33.

Martinez N J, Walhout A J (2009) The interplay between transcriptionfactors and microRNAs in genome-scale regulatory networks. Bioessays 31:435-445.

Megiorni F, Cialfi S, Dominici C, Quattrucci S, Pizzuti A (2011)Synergistic post-transcriptional regulation of the Cystic FibrosisTransmembrane conductance Regulator (CFTR) by miR-101 and miR-494specific binding. PLoS One 6: e26601.

Nuzzo F, Radu C, Baralle M, et al. Antisense-based RNA therapy of factorV deficiency: in vitro and ex vivo rescue of a F5 deep-intronic splicingmutation. Blood 2013; 122:3825-31.

Oglesby I K, Chotirmall S H, McElvaney N G, Greene C M (2013) Regulationof cystic fibrosis transmembrane conductance regulator by microRNA-145,-223, and -494 is altered in DeltaF508 cystic fibrosis airwayepithelium. J Immunol 190: 3354-3362.

Ramachandran S, Karp P H, Jiang P, Ostedgaard L S, Walz A E, et al.(2012) A microRNA network regulates expression and biosynthesis ofwild-type and DeltaF508 mutant cystic fibrosis transmembrane conductanceregulator. Proc Natl Acad Sci USA 109: 13362-13367.

Ramachandran S, Karp P H, Osterhaus S R, Jiang P, Wohlford-Lenane C, etal. (2013) Post-transcriptional Regulation of CFTR Expression andFunction by MicroRNAs. Am J Respir Cell Mol Biol.

Ramalho A S, Beck S, Meyer M, Penque D, Cutting G R and Amaral M D. Fivepercent of normal cystic fibrosis transmembrane conductance regulatormRNA ameliorates the severity of pulmonary disease in cystic fibrosis.Am J Respir Cell Mol Biol 2002; 27:619-27.

Ramsey B W, Davies J, McElvaney N G, Tullis E, Bell S C, Dřevinek P,Griese M, McKone E F, Wainwright C E, Konstan M W, Moss R, Ratjen F,Sermet-Gaudelus I, Rowe S M, Dong Q, Rodriguez S, Yen K, Ordoñez C,Elborn J S; VX08-770-102 Study Group. A CFTR potentiator in patientswith cystic fibrosis and the G551D mutation. N Engl J Med. 2011 Nov. 3;365(18):1663-72.

Rene C, Lopez E, Claustres M, Taulan M and Romey-Chatelain M C.NF-E2-related factor 2, a key inducer of antioxidant defenses,negatively regulates the CFTR transcription. Cell. Mol. Life Sci. 2010;67:2297-2309.

Romey M C, Pallares-Ruiz N, Mange A, Mettling C, Peytavi R, et al.(2000) A naturally occurring sequence variation that creates a YY1element is associated with increased cystic fibrosis transmembraneconductance regulator gene expression. J Biol Chem 275: 3561-3567.

Saint-Criq V, Ruffin M, Rebeyrol C, et al. Azithromycin fails to reduceinflammation in cystic fibrosis airway epithelial cells. Eur J Pharmacol2012; 674: 1-6.

Sawicki G S, Sellers D E, Robinson W M. High treatment burden in adultswith cystic fibrosis: challenges to disease self-management. J CystFibros. 2009 March; 8(2):91-6.

Shalgi R, Brosh R, Oren M, Pilpel Y, Rotter V (2009) Couplingtranscriptional and post-transcriptional miRNA regulation in the controlof cell fate. Aging (Albany N.Y.) 1: 762-770.

Sinn P L, Anthony R M, McCray P B, Jr. (2011) Genetic therapies forcystic fibrosis lung disease. Hum Mol Genet 20: R79-86.

Sun G, Li H, Rossi J J (2010) Sequence context outside the target regioninfluences the effectiveness of miR-223 target sites in the RhoB 3′UTR.Nucleic Acids Res 38: 239-252.

Van Goor F, Hadida S, Grootenhuis P D, Burton B, Stack J H, Straley K S,Decker C J, Miller M, McCartney J, Olson E R, Wine J J, Frizzell R A,Ashlock M, Negulescu P A. Correction of the F508del-CFTR proteinprocessing defect in vitro by the investigational drug VX-809. Proc NatlAcad Sci USA. 2011 Nov. 15; 108(46):18843-8.

Verkman A S, Galietta L J. Chloride channels as drug targets. Nat RevDrug Discov. 2009 February; 8(2):153-71.

Varambally S, Cao Q, Mani R S, Shankar S, Wang X, et al. (2008) Genomicloss of microRNA-101 leads to overexpression of histonemethyltransferase EZH2 in cancer. Science 322: 1695-1699.

Viart V, Des Georges M, Claustres M, et al. Functional analysis of apromoter variant identified in the CFTR gene in cis of a frameshiftmutation. Eur J Hum Genet 2012; 20: 180-184.

Webb T R, Parfitt D A, Gardner J C, et al. Deep intronic mutation inOFD1, identified by targeted genomic next-generation sequencing, causesa severe form of X-linked retinitis pigmentosa (RP23). Hum Mol Genet2012; 21:3647-54.

The invention claimed is:
 1. A synthetic or recombinant oligonucleotidecomprising a nucleic acid sequence selected from the group consisting ofi) SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO:5 or ii) RNA encoded by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ IDNO: 4 and SEQ ID NO: 5, wherein the synthetic or recombinantoligonucleotide includes at least one chemical modification.
 2. Apharmaceutical composition comprising the synthetic or recombinantoligonucleotide according to claim 1 and a pharmaceutical acceptablecarrier.
 3. The synthetic or recombinant oligonucleotide of claim 1,wherein the at least one chemical modification is a backbonemodification, a heterocycle modification, a sugar modification ormodification by conjugation.
 4. The synthetic or recombinantoligonucleotide of claim 1, wherein the at least one chemicalmodification increases clinical efficacy and/or in vivo resistance todegradation.
 5. The synthetic or recombinant oligonucleotide of claim 1wherein the oligonucleotide is: a Locked Nucleic Acid (LNA)oligonucleotide, a phosphorodiamidate morpholino oligomer (PMO), a2′-O-Met oligomer, a tricyclo (tc) DNA antisense oligonucleotide (ASO),a U7 short nuclear (sn) RNA, or a tricycle-DNA-oligoantisenseoligonucleotide, or a conjugate of any of these.
 6. The synthetic orrecombinant oligonucleotide of claim 5, wherein the ASO is a U7 mediatedASO or a U1 mediated ASO.
 7. The synthetic or recombinantoligonucleotide of claim 1, wherein the oligonucleotide is apeptide-conjugated oligonucleotide or a nanoparticle-complexedoligonucleotide.
 8. The pharmaceutical composition of claim 2, furthercomprising one or more anti-cystic fibrosis agents.
 9. Thepharmaceutical composition of claim 8, wherein the one or moreanti-cystic fibrosis agents is VX-770, VX-661 or VX-809.
 10. A vectorcomprising an oligonucleotide sequence comprising a nucleic acidsequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, and a heterologousregulatory region.
 11. The vector of claim 10, wherein the vector is anaked plasmid, a phagemid or a viral vector.
 12. The vector of claim 11,wherein the viral vector is a retrovirus vector, a harvey murine sarcomavirus vector, a murine mammary tumor virus vector, and rous sarcomavirus vector; an adenovirus vector, and adeno-associated virus vector;and SV40-type virus vector; a polyoma virus vector, and Epstein-Barrvirus vector, a papilloma virus vector, a herpes virus vector, avaccinia virus vector or a polio virus vector.